A study carried out by the University of Bonn and the University Hospital Bonn throws light on how an important receptor can be inhibited

The human P2X4 receptor plays an important role in chronic pain, inflammation and some types of cancer. Researchers at the University of Bonn and the University Hospital Bonn (UKB) have now discovered a mechanism that can inhibit this receptor. The results were recently published in the scientific journal Nature Communications and open up a pathway for the development of new drugs.

The P2X4 receptor sits in the membrane of many cells. The cell membrane surrounds the cell like a thin skin. The receptor acts as a sort of doorway that is normally closed. It has a latch on the outside that can only be pushed down by a very particular molecule – ATP. If this happens, the receptor opens and allows calcium and sodium ions to flow into the cells, changing how they behave. Certain immune cells with a P2X4 receptor, for example, are activated by ATP and call for assistance from other parts of the body’s defense system. This results in inflammation. In contrast, some nerve cells generate pain stimuli when they are activated in this way.

“This receptor is often overactive in conditions such as chronic inflammation or chronic pain,” explains Prof. Dr. Christa Müller, Head of Pharmaceutical & Medicinal Chemistry at the University of Bonn. “The same is true of some tumor cells – which are driven to keep dividing by ATP and can thus also form metastases.” Pharmaceutical companies around the world are searching for substances that can block the receptor or at least make it less sensitive. However, only very few molecules that can do this have been found up to now. One of them is the anthraquinone derivative PSB-0704 (PSB stands for Pharmaceutical Sciences Bonn), which was developed by Müller’s research group. “We wanted to find out what it actually does and at the same time use this knowledge to help in the development of better drugs,” says Müller, who is also a member of the transdisciplinary research areas (TRA) “Life & Health,” “Matter” and “Sustainable Futures” at the University of Bonn.

Snap-frozen molecules

Her research group has been developing structural biology methods for this purpose over the last few years. However, they had previously been unable to crystallize the receptor together with the inhibitor so that they could understand the structure of the binding state. “That’s why we’ve been using a special method called cryo-EM (cryogenic electron microscopy) instead,” explains the lead author of the publication, Dr. Jessica Nagel, who recently accepted a postdoc position in the USA. “For this method, we produced a solution of the P2X4 receptor and the anthraquinone derivative PSB-0704 and then snap froze it. The resulting film of ice contains millions of receptor molecules together with the bound inhibitor, which we can examine under an electron microscope.”

Nagel and Müller cooperated with researchers at the University Hospital Bonn to analyze the data, in particular with private lecturer Dr. Gregor Hagelüken. The Institute of Structural Biology at the University Hospital Bonn has a lot of experience in finding out how molecules interact with one another. As the molecular complexes lie in different orientations within the ice, it is possible to view them from different angles under the microscope. “We can produce a detailed 3D image by combining these views using special software,” explains Hagelüken.

This method enabled the research group to identify the sites at which the inhibitor docks to the receptor and what impact this has. “When the inhibitor bonds, it causes parts of the P2X4 molecule to move so that it is no longer possible to open the ion channel,” explains Müller’s former doctoral candidate Jessica Nagel, who carried out the majority of the research. This means that the door remains closed even if ATP docks with the receptor.

A molecular “rubber band” makes the binding pocket smaller

PSB-0704 inhibits the opening of P2X4. However, the substance does not do this particularly well and only starts to have an inhibiting effect at relatively high concentrations. The researchers now understand why this is the case: The substance binds within a pocket in the receptor that is quite small and the PSB-0704 molecule does not fit into it very well. This is due to a sort of molecular “rubber band” that pulls the pocket together. “We have developed a receptor without this rubber band,” says Nagel. “And the PSB-0704 inhibitor was almost 700 times more potent as a result.”

This result provides new insights that will help design better drugs. “On the one hand, we can try to design drugs that cut through the molecular rubber band before they bind with the P2X4 receptor,” explains Müller. “An alternative would be to search for smaller molecules that can fit more easily into the binding pocket.”

Her research group has already been working on this subject for a long time: One of her former members of staff Dr. Stephanie Weinhausen started the search for an inhibitor with the support of the computer expert Dr. Vigneshwaran Namasivayam more than ten years ago and laid the foundations for their latest success. The recently published results give cause for hope that it will be possible to develop new drugs that can more effectively inhibit the opening of the P2X4 receptor in the medium term. However, Müller emphasizes that there is still a long road ahead. “Nevertheless, our joint study has now provided the basis upon which we could successfully achieve this goal.”

Participating institutes and funding:

Alongside the University of Bonn and the University Hospital Bonn, the other participants in the study were LMU Munich and the company Cube Biotech in Monheim. The research was funded by the German Research Foundation (DFG), the Federal Ministry of Research, Technology and Space (BMFTR) and the German Academic Exchange Service (DAAD).